Field of the Invention
The preferred embodiments are directed to an apparatus and method of performing force measurements, and more particularly, an improved probe microscope for sensing tip-sample interaction forces that is able to provide real-time discrimination between tip-sample forces of interest and false force signals, i.e., deflection artifacts in typical force spectroscopy or force volume experiments.
Description of Related Art
Force Spectroscopy refers to a measurement where probe sample distance varies in a controlled way by approaching a probe to the sample and retracting from the sample. The interaction force or a related observable is monitored throughout the process. The force as the function of tip-sample distance during approaching and retracting is referred to as force spectroscopy or force curve. Force spectroscopy has long been a key method used by researchers to study a wide range of samples using a wide range of related techniques from pulling (where molecules are stretched and unfolding or binding forces are observed) to indentation (where a probe is pressed into a surface and elastic, plastic or creep properties of the sample are observed) to scratching (where the probe is pressed into the sample and then moved laterally to study wear and coating adhesion). For each of these sub-techniques dedicated instruments have been developed such as optical tweezers or magnetic beads for pulling, dedicated nanoindenters for indentation and automated scratch testers.
In this regard, developments in nanotechnology have enabled mechanical experiments on a broad range of samples including single molecules, such that fundamental molecular interactions can be studied directly. With a force sensitivity on the order of a few pico-Newtons (pN=10−12 N), a particular type of scanning probe microscope (SPM) called an atomic force microscope (AFM) provides an excellent tool for probing fundamental force interactions between surfaces. AFM has been used to probe the nature of the forces between the probe and the sample for many types of interaction forces (van der Waals and electrostatic forces to name two) and has the advantage that there is no requirement that the tip or sample be conducting in order for the technique to work. Some examples of insulating and conducting samples that have been studied include materials such as silicon nitride, diamond, alumina, mica, glass, graphite, and various organic materials. Other applications include the study of adhesion, friction, and wear, including the formation or suppression of capillary condensation on hydrophilic silicon, amorphous carbon and lubricated SiO2 surfaces.
For biological molecules, force is often an important functional and structural parameter. Biological processes such as DNA replication, protein synthesis, and drug interaction, to name a few, are largely governed by intermolecular forces. However, these forces are extremely small. With its sensitivity in the pico-Newton scale, the SPM has been employed to analyze these interactions. In this regard, SPMs typically are used to generate force curves that provide particularly useful information for analyzing very small samples (as small as individual molecules) or larger samples with a high level of detail.
The knowledge regarding the relation between structure, function and force is evolving and therefore single molecule force spectroscopy, particularly using SPM, has become a versatile analytical tool for structural and functional investigation of single bio-molecules in their native environments. For example, force spectroscopy by SPM has been used to measure the binding forces of different receptor-ligand systems, observe reversible unfolding of protein domains, and investigate polysaccharide elasticity at the level of inter-atomic bond flips. Moreover, molecular motors and their function, DNA mechanics and the operation of DNA-binding agents such as proteins in drugs have also been observed. Further, the SPM is capable of making nano-mechanical measurements (such as elasticity) on biological specimens, thus providing data relative to subjects such as cellular and protein dynamics.
Another main application of AFM force measurements is in materials science where the study of mechanical properties of nano-scale thin films and clusters is of interest. For example, as microstructures such as integrated circuits continue to shrink, predicting the mechanical behavior of thin films from known properties of the bulk materials becomes increasingly inaccurate. Therefore, continuing demand for faster computers and larger capacity memory and storage devices places increasing importance on understanding nano-scale mechanics of metals and other commonly used materials.
To understand the challenges associated with these experiments using AFM, it is instructive to review the AFM itself. AFMs are devices that typically use a sharp tip and low forces to characterize the surface of a sample down to atomic dimensions. In addition to surface characteristic imaging such as topographical imaging, the AFM can probe nano-mechanical and other fundamental properties of samples and their surfaces. Again, AFM applications extend into applications ranging from measuring colloidal forces to monitoring enzymatic activity in individual proteins to analyzing DNA mechanics.
In AFM, the probe tip is introduced to a surface of a sample to detect changes in the characteristics of the sample. Relative scanning movement between the tip and the sample is provided so that surface characteristic data can be acquired over a particular region of the sample, and a corresponding map of the sample surface can be generated. Notably, SPMs also include devices such as molecular force probes (MFPs) that similarly use a probe to characterize sample properties, but do not scan.
In one application of AFM, either the sample or the probe is translated up and down relatively perpendicularly to the surface of the sample in response to a signal related to the motion of the cantilever of the probe as it is scanned across the surface to maintain a particular imaging parameter (for example, to maintain a set-point oscillation amplitude). In this way, the feedback data associated with this vertical motion can be stored and then used to construct an image of the sample surface corresponding to the sample characteristic being measured, e.g., surface topography. Other types of images are generated directly from the detection of the cantilever motion or a modified version of that signal (i.e., deflection, amplitude, phase, friction, etc.), and are thus are often able to provide complementary information to topographical images.
A key element of the AFM is the probe. The probe consists of a microscopic cantilever of typical length 10-1000 microns and spring constant of 0.001-1000 N/m. The cantilever is fixed at its base and usually interacts with the sample through a tip located near its free end. To localize the measurement, the AFM probe often has a very sharp tip apex (less than a few nanometers in diameter). These sharp tips allow high resolution mapping of topography (often to choose a region of interest for force spectroscopy) and material properties by scanning laterally across the surface, but are more fragile than larger tips. The range of force that can be applied or observed typically depends on the stiffness (spring constant) of the cantilever to which the tip is attached. To access different ranges of force, the user needs only to change the probe.
A second key element of the AFM is the micropositioner or scanner, which allows the relative position between the base of the cantilever and the sample to be controlled. The relative position of tip and sample can be controlled by either moving the probe, the sample, or some combination of the two. Most AFM scanners allow control of the relative tip-sample position in three dimensions (note that Z-axis scanner may be separate or integral with the AFM scanner providing raster scanning), both approximately perpendicular to the sample surface and approximately parallel to it.
In a typical force spectroscopy ramping operation, the tip is moved relative to the sample surface (usually toward the surface), until a certain force or deflection trigger threshold is met, at which point the system automatically takes an action such as changing the direction or speed of motion. Alternatively, some other measured variable (amplitude, phase, deflection, current, deformation, lateral force, etc.) can be used instead of force and “Z” and/or another system controllable parameter may be adjusted (ramp at a different rate, move laterally to scratch, apply an electrical bias to tip or sample, change the drive amplitude or frequency, etc.) Notably, the wide range of force (a few pN to a few μN) that can be applied with AFM allows it to be adopted for all of these techniques. Moreover, AFM based force spectroscopy can be carried out on conductive or non-conductive samples in air, liquid, vacuum, and over a wide range of temperature. These characteristics have allowed it to be adopted for studies from the nature of intermolecular forces such as van der Waals and molecular folding to adhesion, friction, wear, plastic creep, viscoelasticity, and elasticity.
As an overview, a simple force curve records the force on the tip of the probe as the tip approaches and retracts from a point on the sample surface. The value of force is indicated by deflection of the probe cantilever. With known spring constant, the cantilever defection can be directly converted to interaction force by Hook's law. A more complex measurement known as a “force volume,” is defined by an array of force curves obtained as described above over an entire sample area. Each force curve is measured at a unique X-Y position on the sample surface, and the curves associated with the array of X-Y points are combined into a 3-dimensional array, or volume, of force data. The force value at a point in the volume is the deflection of the probe at that position (x, y, z).
Turning to FIGS. 1A-1E and 2, a typical force curve resulting from force spectroscopy using SPM (AFM) is illustrated. More particularly, FIGS. 1A-1E show how the forces between a tip 14 of a probe 10 and a sample 16, at a selected point (X,Y) on the sample, deflect a cantilever 12 of probe 10 as the tip-sample separation is modulated in a direction generally orthogonal to the sample surface. FIG. 2 shows the magnitude of the forces as a function of sample position, i.e., a force curve or profile.
In FIG. 1A, probe 10 and sample 16 are not touching as the separation between the two is narrowed by moving the sample generally orthogonally toward the sample surface. Zero force is measured at this point of the tip-sample approach, reflected by the flat portion “A” of the curve in FIG. 2. Next, probe 10 may experience a long range attractive (or repulsive force) and it will deflect downwardly (or upwardly) before making contact with the surface. This effect is shown in FIG. 1B. More particularly, as the tip-sample separation is narrowed, tip 14 may “jump” into contact with the sample 16 if it encounters sufficient attractive force from the sample. In that case, the corresponding bending of cantilever 12 appears on the force profile, as shown in FIG. 2 at the curve portion marked “B.”
Turning next to FIG. 1C, once tip 14 is in contact with sample 16, the cantilever will return to its zero (undeflected) position and move upwardly as the sample is translated further towards probe 10. If cantilever 12 of probe 10 is sufficiently stiff, the probe tip 14 may indent into the surface of the sample. Notably, in this case, the slope or shape of the “contact portion” of the force curve can provide information about the elasticity of the sample surface. Portion “C” of the curve of FIG. 2 illustrates this contact portion.
In FIG. 1D, after loading cantilever 12 of probe 10 to a desired force value, the displacement of the sample 16 is reversed. As probe 10 is withdrawn from sample 16, tip 14 may either directly adhere to the surface 16 or a linkage may be made between tip 14 and sample 16, such as via a molecule where opposite ends are attached to the tip 14 and surface 16. This adhesion or linkage results in cantilever 14 deflecting downwards in response to the force. The force curve in FIG. 2 illustrates this downward bending of cantilever 14 at portion “D.” Finally, at the portion marked “E” in FIG. 2, the adhesion or linkage is broken and probe 10 releases from sample 16, as shown in FIG. 1E. Particularly useful information is contained in this portion of the force curve measurement, which contains a measure of the force required to break the bond or stretch the linked molecule.
The maximum force in FIG. 2 is the most important feature in the measurement operation. Once a pre-defined maximum force is reached, also called trigger force or trigger threshold or simply trigger, the piezo actuator will pull the tip away from the sample and perform the retract measurement, as shown in curve D of FIG. 2. Practically, curve C (approaching) and D (retracting) in FIG. 2 should overlap. Separation is only shown for ease of viewing. The level of the trigger force determines the level of the measurement system performance. State of art instrumentation can operate and a trigger force of a few hundred pN to 1 nN reliably. Lower trigger force is desired in force spectroscopy measurements. Practically, AFM measures deflection and converts deflection to force. The trigger force is represented by a predefined deflection value. When the probe is not interacting with the sample, the deflection remains constant. Variation of deflection relative to the constant is generally caused by tip sample interaction and is used as a measure of the tip-sample interaction force.
Although SPMs are particularly useful in making the above-described measurements, there have been problems with such systems. Experimentally, in the example shown in FIGS. 1A-E and 2, a force curve measurement is made by applying, for example, a cyclical triangle wave voltage pattern to the electrodes of the Z-axis scanner. Such conventional systems often lack flexibility in making measurements that are non-cyclic. The triangle wave drive signal causes the scanner to expand and then contract in the vertical direction, generating relative motion between the probe and the sample. In such a system, the amplitude of the triangle wave as well as the frequency of the wave can be controlled so that the researcher can linearly vary the distance and speed that the AFM cantilever tip travels during the force measurement.
Oftentimes it is desired to modify the parameters of the force measurement in a non-cyclical manner, including the speed at which the tip-sample separation is modulated, the duration of a pause (to allow molecular binding between tip and molecules on the surface, for example), etc. to analyze forces corresponding to, for example, complex mechanical models of certain samples. In U.S. Pat. Nos. 6,677,697 and 7,044,007 assigned to Bruker Nano, Inc., each of which is expressly incorporated by reference herein, a system and method are disclosed in which the flexibility in performing the force measurement is improved. For example, a specific change or rate of change in tip-sample force or a specific value of a tip-sample force may indicate some property pertaining to the sample in question. In response, the instrument alters a force curve measurement parameter (such as the speed of the movement) in response to a specific measurement condition. Or, for example, rather than following a path of position (separation) versus time, the system is able to follow a path of force versus time where the position (separation) is controlled to produce the desired force profile.
Nonetheless, drawbacks still persisted. One in particular has been the ability to factor in the background (or baseline force) while making such measurements. FIG. 3 schematically illustrates deflection associated with, for example, the force curve of FIG. 2, including the effect of a deflection artifact, due to factors described herein that are not related to actual interaction between the probe tip and sample. As shown, the baseline of the force curve, as the probe and sample are brought in to contact from a position in which there is no tip-sample interaction, has a non-ideal slope “S”. As described previously, this could be caused by many factors including system drift, etc. When attempting to trigger operation of the force measurement based on particular tip-sample force(s) this deflection artifact can make it appear that the threshold trigger force has been achieved when in fact it has not, clearly a problem when attempting to measure pico-scale forces.
Stepping back, previously the force trigger was either an absolute threshold, or a relative threshold based on the background deflection when the tip is at its ramp start position. Ideally, the absolute trigger would be sufficient to address most experiment types; however, it has been realized that using a “relative threshold” would simplify operation when the system is not perfectly aligned or if there was some long term drift of the cantilever deflection. Relative triggering is preferred in most cases where the ramp begins with the tip far enough from the sample that it is unaffected by forces due to interaction with the sample. Unfortunately, this does nothing to address the situation where the measured cantilever deflection changes (during the ramp) do not arise from a force between the sample and the tip, but instead result from a measurement “artifact”. Stated another way, absolute triggering works best when there is no measurement artifact (the measurement baseline=0), and relative triggering works best when there is a constant offset to the artifact (baseline=constant). If the baseline is not constant during the ramp, it is clear that the real deflection and force can be either smaller or larger than the trigger threshold. Since the “false deflection” (deflection before the tip interacts with the surface) can affect the trigger, the precision, the repeatability, and fine control of the deflection trigger, the baseline variation has been a major concern for both AFM makers and users.
To improve the precision and repeatability of the real-time trigger in force spectroscopy, AFM manufacturers have attempted to design systems to reduce the false deflection. However, eliminating the false deflection due to imperfections in the optical path and cantilever base motion (see FIG. 4A), cantilever bending that is unrelated to the force on the tip, e.g., thermal effects causing the tip to bend downwardly with temperature change (see FIG. 4B), and presence of light scattered from the sample (FIG. 4C—the top view of a probe 50 on the left shows a laser light spot 52 from a beam L filling the width of the lever, with some light L′ spilling laterally over the sides of the lever and on to the sample 54), or interference due to non-plenary cantilever surface, has remained a challenge. Data illustrating two such deflection artifacts are shown in FIG. 5A (deflection artifact due to optical interference and imperfect optical path causing a positive slope and modulation 40 in the force curve) and 5B (deflection artifact due to strong optical interference 44 in force curve 42, showing both large positive and negative slope in the measured deflection). Notably, as developers turn to shorter cantilevers for higher bandwidth low noise measurements, some of these effects are exacerbated.
Previous attempts to address this issue by processing the data have primarily been confined to offline analysis, where algorithms have been applied to correct the force spectra after acquisition. The algorithms typically calculate the baseline slope (from the part of the curve where the tip is not yet interacting with the surface, e.g., approach) and subtract it from the dataset. Once the artifact in the data is corrected by removing slope in the deflection measurement when the tip is not in proximity to the sample (distance larger than 10 nm), the real maximum force or trigger force can be recovered. The corrected data, as a whole, for both approaching and retracting are then used to derive the sample properties in further analysis. This partially addresses the problem, but ignores the fact that the tip and sample can be altered by the history of force that they experience. There are at least three distinctive cases in which the measurement may be adversely impacted. In one case, the slope or variation of deflection artifact can reach the predefined trigger force (or trigger threshold for deflection). In that case, the piezo actuator of the AFM system will retract the probe based on this false trigger.
As a result, the force spectroscopy data thereby acquired would not reflect any tip sample interaction. This is commonly known as a false triggered force curve. In another case, and particularly when the slope is tilting downwardly as the probe approaches the sample (further illustrated in FIG. 18), the maximum trigger force can be substantially higher than the desired trigger force. The sample or the probe can be irreversibly damaged. In the third case, multiple force curves may need to be measured at an identical trigger force. In this case, artifact deflection can modify the individual trigger due to time or positional variation of the artifact between each set of multiple force curves. The big challenge has been how to discriminate the artifact from the important data before the whole ramp is collected. In that case, the system could automatically take action based on the artifact free data to change the ramp direction, or velocity, apply a bias to tip or sample, or adjust another system controllable parameter.
An example of real-time false deflection correction for AFM is suggested in U.S. Pat. No. 8,650,660, assigned to Bruker Nano, Inc. (the entirety of which is expressly incorporated by reference herein), which discloses the use of the so-called Peak Force Tapping mode to perform mechanical property measurements. In this case, the assumption is made that the false deflection is “nearly constant” and the system lifts the probe to measure the false deflection artifact with no interaction and then subtracts the measured artifact from all subsequent curves. Unfortunately, the false deflection can vary over time and as a function of position. In particular, the false deflection can be very sensitive to distance between the tip and sample, making it impractical to use this technique without first finding the surface by touching it with the tip. This rules out this method for experiments where the first contact of the tip and sample must be observed, or when the tip is particularly fragile and the tip-sample force must never exceed a value that is less than the deflection background. Additionally, the assumption that the false deflection is constant often fails when the force measurements are separated laterally by more than a few tens of nanometers.
Real-time discrimination was desired because it (1) allows the tip or functionalized groups on the tip to be preserved, (2) allows data to be collected while the sample is in a specific configuration that could be disrupted by any further change in force (through irreversible deformation or detachment of a molecule from the tip), and (3) allows the system to perform other actions (scratch, hold, change direction of motion) once a specific force is reached.